天然低共熔溶剂中α-L-鼠李糖苷酶催化黄烷酮糖苷脱糖基化反应的研究

Glycosylated flavanones reprensented by hesperidin and naringin is difficult to be hydrolyzed in vitro because of their low solubility, which is troubling enzymatic turbidity preventing and debittering methods in citrus industry. Deglycosylation of flavanone glycosides catalyzed α-L-rhamnosidase is selected as the model reaction because it is a key protocol of enzymatic preventing turbidity and debittering methods in citrus industry.A kind of new green medium, called as natural deep eutectic solvent (NADES), combined the enhanced solubility of flavanone glycoside and high enzymatic activities will be designed and synthesized by using natural small organic molecules which are abundant in plant cells. The dissolution characteristics and molecular mechanism of flavanone glycoside in NADES will be investigated. Combined with the composition and physical & chemical properties of NADES, and with its effect on the solubility, the reaction equilibrium and the molecular structure of enzyme in the reaction system, the molecular basis of the effect of NADES on the enzyme will be revealed. In this project, a green extraction process with NADES for flavanone glycosides and a green, efficient and highly selective biotransformation system for flavanone glycosides in vitro will be established, which is hopeful to improve the efficiency of enzymatic debittering and turbidity preventing in citrus industry and provide technical support for making profit from the waste and whole fruit processing. If the system is slightly modified , it can be used to dissolve and biotransform the other insoluble compound.

柑橘中以橙皮苷和柚皮苷为代表的黄烷酮糖苷因溶解度低而体外难以被水解，限制酶法防浊脱苦技术在柑橘工业的发展。本项目以酶法防浊脱苦的关键反应——α-L-鼠李糖苷酶催化黄烷酮糖苷脱糖基化反应为模型反应，利用植物细胞内存在的大量天然有机小分子设计并合成对黄烷酮糖苷具有高溶解度而又跟酶相容的天然低共熔溶剂（Natural Deep Eutectic Solvent, NADES)，探究黄烷酮糖苷在NADES中的溶解特性及分子机理；结合其组成和物化性质、酶解反应特性和酶分子结构，探讨NADES对α-L-鼠李糖苷酶促黄烷酮糖苷脱糖基化反应影响的分子基础。本项目建立的柑橘黄酮类化合物NADES绿色提取工艺和绿色高效高选择性黄烷酮糖苷体外生物转化体系，可望提高酶法防浊脱苦效率和柑橘产业加工水平，为柑橘下脚料变废为宝和全果加工提供技术支撑。研究具有一定通用性，稍加改造，也可用于溶解和生物转化其它难溶物。

柑橘中的黄烷酮糖苷含量非常丰富，如何充分的利用一直是科研工作者们努力的方向。.项目主要从以下几个方面进行了研究：.1）利用植物细胞内存在的大量天然有机小分子设计并合成、挑选对黄烷酮糖苷具有高溶解度而又跟酶相容的天然低共熔溶剂NADESs，分别建立 NADESs 绿色提取柑橘黄酮类化合物如橙皮苷、柚皮苷和茶碱等工艺，经此工艺提取的柚皮脱苦效果显著。 .2）建立一个高效、高选择性的酶解黄烷酮糖苷体系。结合NADESs组成和物化性质、 酶解反应特性和酶分子结构，通过分子对接和动力学模型探讨NADES对α-L-鼠李糖苷酶促黄烷酮糖苷脱糖基化反应影响的分子基础。氯化胆碱甘油组成的NADESs有助于活性中心的Asp335和Glu595残基催化橙皮苷的α-1,6糖苷键的水解。氯化胆碱L-脯氨酸组成的NADESs有助于活性中心的Arg37和Glu39催化柚皮苷的α-1,2糖苷键水解。.3）提取和水解黄烷酮糖苷体系的应用研究：a)提取的柚皮苷为前体物质合成了一种性能优良的蓝色荧光CDs，并成功将CDs用于Al3+的检测。b）酶解黄烷酮糖苷副产物β-D-G构建功能性变色水凝胶。c）开发一种高效高灵敏的检测茶碱生物传感方法.本项目解决了黄烷酮糖苷溶解度低、体外难以生物转化的难题；经过天然共熔绿色溶剂提取后的柚皮苦味明显降低，以及柚皮苷和水解副产物β-D-G的进一步开发利用，为柑橘加工业下脚料变废为宝提供了广大空间，有望提高柑橘加工产业的总体水平。